Detergent enzymes have been marketed for more than 20 years and are today well established as normal detergent ingredients in both powder and liquid detergent all over the world.
Detergent compositions may comprise many different enzymes, of which proteases, amylases, cellulases, lipases, cutinases are the most important today. In this context lipolytic enzymes serve to remove lipid or faty stains from clothes and other textiles.
Various microbial lipases have been suggested as detergent enzymes. Examples of such lipases include a Humicola lanuginosa lipase, e.g. described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, e.g. as described in EP 238 023 and Boel et al., Lipids 23, 701–706, 1988, Absidia sp. lipolytic enzymes (WO 96/13578), a Candida lipase, such as a C. antarctica lipase, e.g. the C. antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g. as described in EP 218 272, a P. cepacia lipase, e.g. as described in EP 331 376, a Pseudomonas sp. lipase as disclosed in WO95/14783, a Bacillus lipase, e.g. a B. subtilis lipase (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253–260), a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422).
Furthermore, a number of cloned lipases have been described, including the Penicillium camembertii lipase described by Yamaguchi et al., (1991), Gene 103, 61–67), the Geotricum candidum lipase (Schimada, Y. et al., (1989), J. Biochem., 106, 383–388), and various Rhizopus lipases such as a R. delemar (R. D. Joerger and M. J. Hass (1993), Lipids 28 p. 81–88), a R. niveus lipase (W. Kugimiya et al. (1992), Biosci. Biotech. Biochem. 5, p. 716–719), R. javinicus (W. Uyttenbroeck et al. (1993) Biol. chem. Hoppe-Seyler 374, p. 245–254) and a R. oryzae (Haas, M. J., Allen, J. and Berka, T. R. (1991) Gene 109, p. 107–113) which has a substantially identical sequence to the other Rhizopus lipases.
Other types of lipolytic enzymes having been suggested as detergent enzymes include cutinases, e.g. derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446).
In recent years attempts have been made to prepare modified lipolytic enzymes, such as variants and mutants having improved properties for detergent purposes.
For instance, WO 92/05249 discloses lipase variants with improved properties, in which certain characteristics of wild-type lipase enzymes have been changed by specific, i.e. site-directed modifications of their amino acid sequences. More specifically, lipase variants are described, in which one or more amino acid residues of the so-called lipid contact zone of the parent lipase has been modified.
WO 94/01541 describes lipase variants with improved properties, in which an amino acid residue occupying a critical position vis a vis the active site of the lipase has been modified.
EP 407 225 discloses lipase variants with improved resistance towards proteolytic enzymes, which have been prepared by specifically defined amino acid modifications.
EP 260 105 describes hydrolases in which an amino acid residue within 15 Å from the active site has been substituted.
WO 95/35381 discloses Pseudomonas sp. lipase variants, in particular P. glumae and P. pseudoalcaligenes lipase variants which have been modified so as to increase the hydrophobicity at the surface of the enzyme.
WO 96/00292 discloses Pseudomonas sp. lipase variants, in particular P. glumae and P. pseudoalcaligenes lipase variants which have been modified so as to improve the enzyme's compatibility to anionic surfactants,
WO 95/30744 discloses mutant lipases such as Pseudomonas sp. lipases which have been modified to an increased surfactant resistance.
WO 94/25578 discloses mutant lipases comprising at least a substitution of the methionine corresponding to position 21 in the P. pseudoalcaligenes lipase, in particular to leucine, serine or alanine.
All of the above mentioned lipase variants have been constructed by use of site-directed mutagenesis resulting in a modification of specific amino acid residues which have been chosen either on the basis of their type or on the basis of their location in the secondary or tertiary structure of the parent lipase.
An alternative approach for constructing mutants or variants of a given protein has been based on random mutagenesis. For instance, U.S. Pat. No. 4,898,331 and WO 93/01285 disclose such techniques.
WO 95/22615 discloses variants of lipolytic enzymes having an improved washing performance, the variants having been prepared by a method involving subjecting a DNA sequence encoding the parent lipolytic enzyme to random mutagenesis and screening for variants having a decreased dependence to calcium and/or an improved tolerance towards a detergent or one or more detergent components as compared to the parent lipolytic enzyme.
WO 95/09909 discloses, inter alia, chemically modified lipases or lipase mutants which has a higher pl than the corresponding modified enzyme.
Comments to Prior Art
It is known from prior art to modify lipolytic enzymes by site-directed mutagenesis to obtain an improved performance, in particular washing performance of lipolytic enzymes. The generally used concept has been to insert, delete or substitute amino acids within the structural part of the amino acid chain of the parent lipolytic enzyme in question. Lipolytic enzymes with a significantly improved washing performance have been achieved this way.
However, there is a need for providing lipolytic enzymes with an even further improved performance, such as washing performance and/or even further improved dishwashing properties than the lipolytic enzymes prepared by these prior art methods.
Furthermore, a drawback of all detergent lipolytic enzymes described until now is that they exert the best fat removing effect after more than one wash cycle, presumably because the known lipolytic enzymes, when deposited on the fatty stain to be removed, are more active during a certain period of the drying process than during the wash process itself (Gormsen et al., in Proceedings of the 3rd World Conference on Detergents, AOCS press, 1993, pp 198–203). This has the practical consequence that at least two wash cycles (separated by a sufficient drying period) are required to obtain a substantial removal of fatty stains.
Some lipolytic enzymes have been described as allegedly being capable of removing fatty matter during the first wash cycle. Thus, WO 94/03578 discloses a detergent composition which in addition to various detergent components an enzyme which is alleged to be capable of exhibiting a substantial lipolytic activity during the main cycle of a wash process. Examples of lipolytic enzymes allegedly exhibiting the above activity include stem-specific cutinases such as the cutinase from Fusarium solani pisi, Fusarium roseum culmorum, Rhizoctonia solani and Alternaria brassicicola. However, when tested under realistic washing conditions none of these enzymes are capable of removing substantial amounts of a fatty stain during a one cycle wash process (cf the examples hereinafter).
Thus, a need exists for lipolytic enzymes which under realistic wash conditions are capable of removing substantial amounts of fatty matter during one wash cycle.